Achievement of a desirable and stably inheritable pattern of transgene expression remains one of the major problems in plant biotechnology. The standard approach is to introduce a transgene as part of a fully independent transcription unit using a vector, where the transgene is under transcriptional control of a plant-specific heterologous or a homologous promoter and transcription termination sequences (for example, see U.S. Pat. No. 05,591,605; U.S. Pat. No. 05,977,441; WO 0053762 A2; U.S. Pat. No. 05,352,605, etc). However, after the integration into the genomic DNA, because of random insertion of exogenous DNA into plant genomic DNA, gene expression from such transcriptional vectors becomes affected by many different host factors. These factors make transgene expression unstable, unpredictable and often lead to the transgene silencing in progeny (Matzke & Matzke, 2000, Plant Mol Biol., 43, 401-415; S. B. Gelvin, 1998, Curr. Opin. Biotechnol., 9, 227-232; Vaucheret et al., 1998, Plant J., 16, 651-659). There are well-documented instances of transgene silencing in plants, which include the processes of transcriptional (TGS) and posttranscriptional gene silencing (PTGS). Recent findings reveal a close relationship between methylation and chromatin structure in TGS and involvement of RNA-dependent RNA-polymerase and a nuclease in PTGS (Meyer, P., 2000, Plant Mol. Biol, 43, 221-234; Ding, S. W., 2000, Curr. Opin. Biotechnol., 11, 152-156; lyer et al., Plant Mol. Biol., 2000, 43, 323-346). For example, in TGS, the promoter of the transgene can often undergo methylation at many integration sites with chromatin structure not favorable for stable transgene expression. As a result, practicing existing methods requires many independent transgenic plants to be produced and analyzed for several generations in order to find those with the desired stable expression pattern. Moreover, even such plants displaying a stable transgene expression pattern through the generations can become subsequently silenced under naturally occurring conditions, such as a stress or pathogen attack. Existing approaches aiming at improved expression control, such as use of scaffold attachment regions (Allen, G. C., 1996, Plant Cell, 8, 899-913; Clapham, D., 1995, J. Exp. Bot., 46, 655-662; Allen, G. C., 1993, Plant Cell, 5, 603-613) flanking the transcription unit, could potentially increase the independency and stability of transgene expression by decreasing dependency from so-called “position effect variation” (Matzke & Matzke, 1998, Curr. Opin. Plant Biol., 1, 142-148; S. B. Gelvin, 1998, Curr. Opin. Biotechnol., 9, 227-232; WO 9844 139 A1; WO 006757 A1; EP 1 005 560 A1; AU 00,018,331 A1). However, they only provide a partial solution to the existing problem of designing plants with a required expression pattern of a transgene.
Gene silencing can be triggered as a plant defence mechanism by viruses infecting the plant (Ratcliff et al., 1997, Science, 276, 1558-1560; Al-Kaff et al., 1998, Science, 279, 2113-2115). In non-transgenic plants, such silencing is directed against the pathogen, but in transgenic plants it can also silence the transgene, especially when the transgene shares homology with a pathogen. This is a problem, especially when many different elements of viral origin are used in designing transcriptional vectors. An illustrative example is the recent publication by Al-Kaff and colleagues (Al-Kaff et al., 2000, Nature Biotech., 18, 995-999) who demonstrated that CaMV (cauliflower mosaic virus) infection of a transgenic plant with the BAR gene under the control of the CaMV-derived 35S promoter can silence the transgene.
During the last years, the set of cis-regulatory elements has significantly increased and presently includes tools for sophisticated spatial and temporal control of transgene expression. These include several transcriptional elements such as various promoters and transcription terminators as well as translational regulatory elements/enhancers of gene expression. In general, translation enhancers can be defined as cis-acting elements which, together with cellular trans-acting factors, promote the translation of the mRNA. Translation in eukaryotic cells is generally initiated by ribosome scanning from the 5′ end of the capped mRNA. However, initiation of translation may also occur by a mechanism which is independent of the cap structure. In this case, the ribosomes are directed to the translation start codon by internal ribosome entry site (IRES) elements. These elements, initially discovered in picornaviruses (Jackson & Kaminski, 1995, RNA, 1, 985-1000), have also been identified in other viral and cellular eucaryotic mRNAs. IRES are cis-acting elements that, together with other cellular trans-acting factors, promote assembly of the ribosomal complex at the internal start codon of the mRNA. This feature of IRES elements has been exploited in vectors that allow for expression of two or more proteins from polycistronic transcription units in animal or insect cells. At present, they are widely used in bicistronic expression vectors for animal systems, in which the first gene is translated in a cap-dependent manner and the second one is under the control of an IRES element (Mountford & Smith, 1995, Trends Genet, 4, 179-184; Martines-Salas, 1999, Curr Opin Biotech., 19, 458-464). Usually the expression of a gene under the control of an IRES varies significantly and is within a range of 6-100% compared to cap-dependent expression of the first one (Mizuguchi et al., 2000, Mol. Ther., 1, 376-382). These findings have important implications for the use of IRESs, for example for determining which gene shall be used as the first one in a bicistronic vector. The presence of an IRES in an expression vector confers selective translation not only under normal conditions, but also under conditions when cap-dependent translation is inhibited. This usually happens under stress conditions (viral infection, heat shock, growth arrest, etc.), normally because of the absence of necessary trans-acting factors (Johannes & Sarnow, 1998, RNA, 4, 1500-1513; Sonenberg & Gingras, 1998, Cur. Opin. Cell Biol., 10, 268-275).
Translation-based vectors recently attracted attention of researchers working with animal cell systems. There is one report connected with the use of an IRES-Cre recombinase cassette for obtaining tissue-specific expression of cre recombinase in mice (Michael et al., 1999, Mech. Dev., 85, 35-47). In this work, a novel IRES-Cre cassette was introduced into the exon sequence of the EphA2 gene, encoding an Eph receptor of protein tyrosine kinase expressed early in development. This work is of specific interest as it is the first demonstration of the use of translational vectors for tissue-specific expression of a transgene in animal cells that relies on transcriptional control of the host DNA. Another important application for IRES elements is their use in vectors for the insertional mutagenesis. In such vectors, the reporter or selectable marker gene is under the control of an IRES, element and can only be expressed if it inserts within the transcribed region of a transcriptionally active gene (Zambrowich et al., 1998, Nature, 392, 608-611; Araki et al., 1999, Cell Mol. Biol., 45, 737-750). However, despite the progress made in the application of IRESs in animal systems, IRES elements from these systems are not functional in plant cells. Moreover, since site-directed or homologous recombination in plant cells is extremely rare and of no practical use, similar approaches with plant cells were not contemplated.
There are significantly less data about plant-specific IRES elements. Recently, however, several IRESs that are also active in plants were discovered in tobamovirus crTMV (a TMV virus infecting Cruciferae plants) (Ivanov et al., 1997, Virology, 232, 32-43; Skulachev et al., 1999, Virology, 263, 139-154; WO 98/54342) and there are indications of IRES translation control in other plant viruses (Hefferon et al., 1997, J. Gen Virol., 78, 3051-3059; Niepel & Gallie, 1999, J. Virol, 73, 9080-9088). IRES technology has a great potential for the use in transgenic plants and plant viral vectors providing convenient alternative to existing vectors. Up to date, the only known application of plant IRES elements for stable nuclear transformation is connected with the use of IRESs to express a gene of interest in bicistronic constructs (WO 98/54342). The construct in question comprises, in 5′ to 3′ direction, a transcription promoter, the first gene linked to the said transcription promoter, an IRES element located 3′ to the first gene and the second gene located 3′ to the IRES element, i.e., it still contains a full set of transcription control elements.
Surprisingly, we have found that translational vectors that are devoid of their own transcription control elements and rely entirely on insertion into a transcriptionally active genomic DNA of a plant host, allow recovery of numerous transformants which express the gene of interest. Even more surprisingly, such transformants could be easily detected even in host plants with a very low proportion of transcriptionally active DNA in their genome such as wheat. This invention is the basis of the proposed process that allows for design of transgene expression that is entirely controlled by the host's transcriptional machinery, thus minimizing the amount of xenogenetic regulatory DNA elements known to trigger transgene silencing. It also allows to control transgene expression in a novel way, by modulating the ratio of cap-dependent versus cap-independent translation.